Abstract
Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.
Highlights
Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands
The underlying cause of CF is the presence of mutations in the CFTR gene that lead to defective functions of the corresponding cystic fibrosis transmembrane conductance regulator (CFTR), a 1480-amino acid transmembrane protein principally located in the apical membrane of cells lining exocrine glands, including lung, sinus, pancreas, intestine, sweat ducts, and vas deferens
blue native (BN)/SDS-PAGE Analysis of Membrane Proteins from Murine Colonic Crypts—The technique of BN-PAGE was initially developed for analysis of the mitochondrial proteome, allowing characterization of many proteins that cannot be resolved using conventional IEF SDS-PAGE
Summary
Chemicals and Animals—All reagents necessary for sample preparation, electrophoresis, and mass spectrometry were of highest grade available. Crypt Isolation and Sample Preparation—Animals were killed by cervical dislocation, and their distal colon was removed and immediately rinsed with cold HEPES-buffered solution (10 mM HEPES, pH 7.2, 140 mM NaCl, 47 mM KCl, 1 mM MgCl2). Gels were fixed in 50% methanol and 10% acetic acid followed by washing three times for 20 min in milli-Q water. For cysteine reduction and alkylation, dried gel pieces were incubated at 56 °C for 45 min in a solution containing 10 mM DTT and 25 mM ammonium bicarbonate. Total membrane protein extracts from crypts of the distal colon were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad) according to the standard protocol. The intensity of visualized bands was semiquantified using the ImageMaster 2D Elite software, version 4.01 (Amersham Biosciences)
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